The perturbation in chromatin structure is one of the most striking and characteristic changes in cancer cells and remains one of the hallmarks in cancer diagnosis. At the molecular level, it is well recognized that aberrant epigenetic marks of DNA methylation and histone modification as well as chromatin organization play an important role in cancer progression, both globally and locally. Despite the importance of epigenetic markers in carcinogenesis, how they influence chromatin structures at the single-cell level in the context of tissue architecture in cancer progression is not well understood. This is in part due to the lack of effective tools to directly visualize and quantify in-situ high-order chromatin structures at a single-cell level in the context of tissue architecture to define cancer phenotype and cell types. Current biochemical assays such as chromatin immunoprecipitation (ChIP) use bulk analysis from millions of cells often with mixed cell types from tissue, and the spatial context of tissue architecture and the original chromatin packaging are lost. Recent advance in super-resolution fluorescence microscopy such as stochastic optical reconstruction microscopy (STORM) is revolutionary by breaking the fundamental resolution limit. It is now possible to visualize the previously invisible high-order chromatin structures in situ down to a resolution of 20-30 nm in a single cell nucleus. Despite its significant impact, it is generally deemed as a complex and low-throughput technique. Our goal is to develop a high-throughput multi-color 3D STORM microscopy system for in-situ visualization of nanoscale chromatin structures defined by different epigenetic marks on patient-derived formalin-fixed paraffin-embedded (FFPE) tissue in their spatial context of tissue architecture in cancer progression. We will implement a large flat-field illumination, various approaches to suppress the background and develop a high-speed high-fidelity 3D image reconstruction algorithm to precisely localize dense overlapping single molecules from heterogeneous background. In our preliminary studies, we have demonstrated the ability to image chromatin structures marked by histone marks and DNA methylation in FFPE tissue and characterized their alteration from human colon cancer progression in a pilot study. During the R33 phase, we will further improve the throughput, develop an automated image acquisition and quantitative analysis platform and characterize alterations of chromatin structures marked by a set of epigenetic marks important in cancer progression. The high- throughput 3D STORM system will significantly advance the widespread use of this powerful imaging tool to directly visualize chromatin structures marked by various epigenetic states on the routinely obtained clinical tissue. It will improve our understanding of cancer biology and eventually lead to better cancer diagnosis, prognosis and treatment.